Role of O ₂ in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

Abstract

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O 2 was used to understand how O 2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547[phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O 2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omegaamino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O 2 , conditions similar to those experienced by N 2 -fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O 2 and cell death.IMPORTANCE Rhizobium leguminosarum, a soil bacterium that forms N 2 -fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O 2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O 2 tensions during growth in soil and N 2 fixation in symbiosis with legumes.KEYWORDS INSeq, nodules, Rhizobium leguminosarum, legumes, nitrogen fixation, Pisum sativum R hizobia are alphaproteobacteria able to form symbioses with legumes, on which they induce development of root nodules. Nodules provide an environment with both a low O 2 concentration and the correct nutrients to enable differentiation of rhizobia into nitrogen (N 2 )-fixing bacteroids (1). Bacteroids fix atmospheric N 2 into ammonium (NH 4 ϩ ), which is secreted to the plant host, and are, in turn, provided with

The Medicago truncatula Vacuolar Iron Transporter-Like proteins VTL4 and VTL8 deliver iron to endosymbiotic bacteria at different stages of the infection process

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Multidisciplinary approaches for studying rhizobium–legume symbioses

“…A mariner transposon was used to examine the core genome of R. leguminosarum bv. viciae (Perry and Yost 2014;Perry et al 2016), as well as to investigate the effects of carbon source and O 2 levels, a key signal during symbiosis, on the fitness of gene disruptions (Wheatley et al 2017). Similarly, a Tn5-derived transposon was used to interrogate the core genome of S. meliloti and to inspect how chromosomal gene phenotypes differ when the extrachromosomal replicons are removed (diCenzo et al 2018a).…”

Modeling population heterogeneity from microbial communities to immune response in cells

“…More recently, shotgun metagenomics enabled the sequencing of the full genome of all organisms present in a sample, providing information on functional roles as well [23]. Furthermore, developments in high-throughput genetic screenings such as mariner transposon insertion sequencing (INSeq) have enabled the systematic study of gene essentiality and function in bacterial populations [24]. With improvement of single-cell RNA preparation [25], and microfluidics devices [26], NGS has been applied to single cell measurements, such as scRNA-seq [27].…”